1. Field of Invention
This invention is directed to compact sensors for detecting the presence of biological materials.
2. Description of Related Art
With the recent attention gained by the possible use of biological agents in committing acts of terrorism, interest has arisen in developing novel methods to quickly detect the presence of very small amounts of biologically active materials. For example, in response to the recent anthrax-by-mail attacks, the US Postal Service is testing systems that collect air samples and test them for the presence of anthrax DNA. The test methodologies include polymerase chain reaction (PCR), wherein an enzyme called a DNA polymerase makes a copy of certain DNA in a chromosome. After the strands of the double-helix of the DNA are separated, the pre-designed primer(s) anchor the target DNA and use it as a template. DNA polymerase makes a copy of the target nucleotide sequence franked by the primers by adding the complementary nucleotides. Each duplication step may take about 1 to a few minutes, and the duplication can be repeated 30 or more times, so that after 30 cycles, one billion copies of the DNA can be made from the original template DNA in 30 min to a few hours. Development in fluorescent real-time detection and efforts to make the PCR instrument portable, such as in product offerings by Cepheid of Sunnyvale, Calif., and Smiths Group PLC, of London, England, have greatly improved the usability of this method in bio-agent detection. However, false positives from contaminants and false negatives from interfering substances in the sample still plague this otherwise sensitive detection method. Cost is another issue as the reagent for real-time PCR is expensive, limiting its use only to the more critical incidences.
Other methods of bio-agent detection use a principle of bio-recognition based on immunogenic reaction similar to that of the antibody-antigen recognition. Traditionally both antigen and antibody are naturally occurring proteins, but there are several variants developed to improve different aspects of this technique. For example, aptamers are synthetic short oligonucleotides that can bind to a target antigen similar to an antibody. In many aspects, it is a more robust alternative to a protein antibody and can be synthesized more cheaply in large scale. These immunogenic detection methods were initially developed for clinical diagnostic purposes and have gained popularity in bio-agent detection as they are amenable to more compact and faster detection. Examples are hand-held assays, or TICKETS, from ANP Technologies of Newark, Del. These TICKETS can be made relatively cheaply, however, their sensitivity may be limited to the visualization method used, i.e. colloidal gold and colorimetric detection.
Several other methods are also used to detect bio-agents without using reagents. Laser induced fluorescence (LIF) looks at the spectra of the constituent amino acids on the bio-agents and compares them against information previously collected in a database to identify the unique signature of a particular agent. LIF is inherently lacking in specificity, although there is work underway to combine multiple spectra from different types of molecules to help pinpoint the identity of the target. Raman spectrometry is also used for a similar purpose in product offerings by ChemImage Corp. of Pittsburgh, Pa. However, a comprehensive library is yet to be established for the possible bio-agents. Furthermore, systems based on these optical techniques remain bulky and non-portable.
Therefore, there is a need for a compact and inexpensive instrument for direct detection of unlabeled bio-molecules. This kind of instrument should require minimal sample preparation and low maintenance. Such tools should also be simple, sensitive, and particularly adept at molecular recognition. Furthermore, the tools should be capable of operating automatically and unattended, and in a highly parallel mode to improve speed and detection specificity. Such an instrument may be used as a laboratory tool, as a clinical diagnostic device, or as a field portable/deployable bio-agent detector.
Vollmer et al. in “Protein detection by optical shift of a resonant microcavity,” (Applied Physics Letters, vol. 80, number 21, pp. 4057-4059) reported the specific detection of unlabeled bio-molecules on a spherical surface (R˜0.15 mm) from the frequency shift of whispering-gallery modes (WGMs). The modes were stimulated in a dielectric sphere immersed in an aqueous environment by means of coupling light evanescently from an eroded optical fiber.
The setup of Vollmer et al. is shown schematically in FIG. 1. The output of wavelength-tunable, distributed feedback laser diode 10, operating at about 1.3 μm, is coupled into an optical fiber 40. The optical fiber 40 is stripped of its cladding along a length 20 of the fiber and etched in hydrofluoric acid, to expose the evanescent field from the fiber 40. The fiber 40 is then held in very close proximity to a dielectric sphere 30, such that light from the fiber 40 is coupled by overlap of the evanescent field from the fiber 40 to the dielectric sphere 30. Light is coupled from the fiber 40 in to the micro sphere and circulates about the equator of the sphere 30, if the wavelength of the light is such that an integral number of wavelengths fit inside the circumference of the sphere 30. Resonant modes of the sphere 30 are detected by a detector 50, by measuring dips in the transmission through the fiber 40. The surface of the sphere is prepared with surface immobilized biotinylated bovine serum albumin (BSA) 11, which binds with streptavidin 12. A shift in a measured dip may occur because of the presence of the layers 11 and 12 on the surface of the sphere, which as a result, alters the effective radius of the sphere. A first shift in the wavelength of the adsorption dip of the transmitted laser light occurs as a result of the adherence of the BSA 11 to the sphere 30, and an additional second shift occurs as a result of the binding of the target molecule streptavidin 12, to the BSA 11 on the sphere 30.
Using a setup similar to that described above and shown in FIG. 1, Vollmer et al. have also demonstrated label-free DNA quantification, as reported in Biophysical Journal, volume 85, September 2003, 1974-1979. Vollmer et al. measured the wavelength shift due to the hybridization of a target DNA after chemically modifying the silica spheres with oligonucleotides. Vollmer et al. calculated that the experimental limit of their detection technique is about 6 picograms of DNA per square millimeter (pg/mm2) of surface density. According to Vollmer et al., the highest sensitivity demonstrated with other methods, for example, with SPR (surface plasmon resonance) was about 10 pg/mm2.
Among the drawbacks of the Vollmer et al. approach is the critical alignment necessary between the fiber 40, and dielectric sphere 30. Adequate coupling from the fiber to the sphere is only accomplished when the fiber is within a distance corresponding approximately to the wavelength of the light. Such well-controlled placement of the parts can only be accomplished with precision positioning devices. Therefore, the setup of Vollmer et al. does not lend itself to a compact, robust biosensor, which can operate unattended in a highly parallel configuration.